Enhanced surface display engineering bacteria for catalytic synthesis of spermidine by whole cell

Abstract

The application belongs to the technical field of synthetic biology, and provides an enhanced surface display engineering bacterium for catalyzing synthesis of spermidine by whole cell. The application provides an Escherichia coli for displaying at least one of homoserine dehydrogenase, carboxyspermidine dehydrogenase and carboxyspermidine decarboxylase; and the Ag43 surface display system is strengthened to catalyze synthesis of spermidine and derivatives thereof by whole cell. The Linker peptide connecting the autotransporter Ag43 and the recombinant protein is optimized, the steric hindrance effect is reduced, and the normal function of the fusion protein structure domain is ensured. The expression of Ag43 is optimized from the aspects of assembly and transmembrane transport of the autotransporter Ag43, and the efficiency of displaying the recombinant protein on the surface of the bacterium is improved. The selected enzyme can realize NAD(P) regeneration, and the requirement for cofactors is reduced. The method for producing spermidine provided by the application has a simple process and easily available raw materials, and has a good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology technology, specifically relating to a method for producing spermidine using genetically engineered bacteria. Background Technology

[0002] Spermidine is a naturally occurring autophagy inducer widely found in all organisms. It participates in vital life processes such as cellular DNA replication, mRNA transcription, and protein translation, as well as various pathophysiological processes including stress response protection and metabolic homeostasis. It possesses diverse biological activities, including anti-tumor, anti-aging, anti-inflammatory, cardiovascular protection, and neuromodulation. Due to its excellent antioxidant, anti-aging, and autophagy-promoting effects, spermidine derivatives have been applied in the pharmaceutical and cosmetic fields, promising to provide consumers with safer and more effective raw materials.

[0003] It has been reported that there are three main biosynthetic pathways for spermidine: the traditional pathway mainly using S-adenosylmethionine decarboxylase (dc-AdoMet) / spermine synthase (SPDSY); the alternative pathway mainly using carboxyspermine dehydrogenase (CASDH) / carboxyspermine decarboxylase (CASDC); and the CAPA pathway mainly using carboxyaminopropylguanidine dehydrogenase (CAPADH) / aminopropylguanidine decarboxylase (CAPADC) / urea hydrolase (APAUH). Current research focuses on using metabolic engineering strategies to modify these three intracellular metabolic pathways to achieve maximum spermidine metabolism throughput. This is combined with strategies such as providing sufficient cofactor supply, modifying transport pathways, promoter overexpression, and optimizing fermentation conditions to further increase spermidine yield. However, the aforementioned traditional spermidine biosynthetic methods are mainly based on intracellular metabolism. Even with modifications to transport pathways, problems such as intracellular accumulation and toxicity due to insufficient transport efficiency of spermidine still exist.

[0004] The inventors' previous research utilized the coupling of NAD(P)-dependent homoserine dehydrogenase (HSD) with NADPH-dependent CASDH to construct a spermidine synthesis pathway with regenerable NADPH cofactor: HSD / CASDH / CASDC. This further addressed the difficulty in obtaining the substrate L-aspartate β-semialdehyde. However, the key enzyme CASDH, when overexpressed intracellularly, produces inclusion bodies due to folding errors. Although this issue has been initially resolved by co-expressing molecular chaperone proteins and screening for CASDH from other sources, the problem of intracellular accumulation after excessive product synthesis remains. Further research revealed that weakening the spermidine uptake system PotABCD and enhancing the expression of the spermidine export protein MdtJI did not significantly increase spermidine yield. Whole-cell catalytic fermentation, primarily involving intracellular metabolism, can significantly increase product synthesis yield after optimizing fermentation conditions, but the low reusability and poor stability of the whole-cell catalyst still hinder further increases in spermidine production. Therefore, there is a need to seek efficient strategies to address the problem of intracellular transport of spermidine and to improve the stability and reusability of whole-cell catalysts.

[0005] Bacterial surface display technology fuses functional enzymes with anchored proteins and uses the anchoring structure to position them on the extracellular surface. The enzymes are then used to display the cells to carry out catalytic processes. Because the catalytic reaction occurs on the cell surface, the generation of inclusion bodies can be avoided and the problem of substrate and product transmembrane transport restrictions can be overcome.

[0006] In recent years, bacterial surface display technology has been widely applied in the production of biosynthetic chemicals, biofuels, biopharmaceutical intermediates, and high-value-added natural products. Researchers have achieved highly efficient whole-cell catalytic synthesis of chemicals such as fatty acid esters, biodiesel, chiral alcohols, and amino acid derivatives by displaying functional enzymes such as lipases, cellulases, alcohol dehydrogenases, transaminases, and carboxylic acid reductases on cell surfaces. This technology can also be further applied to numerous fields, including protein or peptide library screening, environmental pollution control, and vaccine development. However, existing surface display systems still face many limitations in industrial applications.

[0007] First, direct fusion of exogenous and anchor proteins often alters the spatial conformation of the fusion protein, affecting the correct folding and exposure of the active site of the target protein. This is particularly true for multi-domain proteins, macromolecular enzymes, and enzymes requiring cofactor binding, where activity is often significantly reduced due to steric hindrance. Second, the direct connection between the target and anchor proteins may interfere with the transmembrane transport of the Ag43 passenger domain, causing some fusion protein to remain in the periplasmic space or form misfolded structures, thus reducing display efficiency. Furthermore, significant differences in the size, structure, and surface charge distribution of enzyme molecules in multi-enzyme cascade systems can easily lead to mutual occlusion, making it difficult for key catalytic sites to contact the substrate and limiting overall catalytic efficiency. Studies have found that linker peptides, as crucial components connecting the target and anchor proteins, play a decisive role in the expression, transport, display efficiency, and catalytic activity of fusion proteins due to their length, flexibility, rigidity, and amino acid composition. Currently, commonly used linker peptides mainly include flexible linker peptides [(GGGGS)n], rigid linker peptides [(EAAAK)n], and hybrid linker peptides formed by combinations of both.

[0008] Secondly, for autotransporters such as Ag43, their surface display efficiency depends not only on the target protein itself but also on the correct assembly process of the outer membrane β-barrel proteins. Numerous studies have shown that the successful localization of autotransporters to the cell surface relies on the β-barrel assembly machinery (BAM complex) to complete the insertion of β-barrel structures and the transmembrane transport of the passenger domain. Most surface display studies focus on optimizing promoters, induction conditions, expression levels, or anchored protein types, with little attention paid to the decisive role of the BAM complex in display efficiency. In fact, when fusion proteins have large molecular weights, complex structures, or form multi-enzyme fusion proteins, the BAM system is prone to decreased assembly efficiency, leading to a large amount of fusion protein remaining in the periplasmic space, misfolding, or degradation by proteases, ultimately resulting in reduced surface display volume and decreased catalytic activity. Therefore, outer membrane assembly capacity has become one of the major bottlenecks limiting efficient surface display. Thus, improving the recognition, transport, and assembly efficiency of heterologous fusion proteins by the BAM complex is an effective strategy to promote improved membrane surface display efficiency. Summary of the Invention

[0009] To address the problems in the prior art, this invention provides a method for surface-displaying cell-catalyzed synthesis of spermidine, which transfers the synthesis via intracellular metabolic pathways to extracellular catalysis, effectively solving the problems of spermidine accumulation and restricted transport within engineered bacteria.

[0010] The present invention also provides an engineered Escherichia coli strain that can display synthetic spermidine on its surface, avoiding inclusion bodies generated by intracellular overexpression of related enzyme systems, and achieving effective soluble expression of related catalytic enzymes.

[0011] To achieve the above objectives, the present invention adopts the following technical solution.

[0012] An Escherichia coli strain capable of displaying an enzyme protein for spermidine synthesis on its surface, the enzyme protein being selected from at least one of homoserine dehydrogenase (HSD), carboxysemidine dehydrogenase (CASDH), and carboxysemidine decarboxylase (CASDC); the enzyme protein is expressed by fusion of a linker peptide with a carrier protein; and the Escherichia coli strain overexpresses an outer membrane protein.

[0013] In some embodiments, the amino acid sequence of the HSD is as shown in SEQ ID NO:1 or 2; the amino acid sequence of the CASDH is as shown in SEQ ID NO:3 or 4; the amino acid sequence of the CASDC is as shown in SEQ ID NO:5; and the amino acid sequence of the Linker peptide is as shown in SEQ ID NO:6.

[0014] In some embodiments, the outer membrane protein is selected from a combination of Bam A, Bam B, Bam C, Bam D, and Bam E, or Omp85. Preferably, the outer membrane protein is Omp85. The amino acid sequence of Omp85 is shown in SEQ ID NO:7, 8, or 9.

[0015] In some embodiments, the carrier protein is an Ag43 autotransporter. Preferably, the Ag43 autotransporter is a full-length or truncated Ag43 autotransporter, and the truncated Ag43 autotransporter includes Ag43-700, Ag-552, Ag43-138, or Ag43-740. The aforementioned truncated Ag43, with reference to YP_026164.1, retains amino acids 1-52 and 700-1039, 1-52 and 552-1039, 1-52 and 138-1039, and 1-52 and 740-1039, respectively. In some embodiments, the amino acid sequence of the Ag43 autotransporter is shown in SEQ ID NO: 10.

[0016] To better express the above proteins, their encoded proteins can be optimized according to the codon preferences of *E. coli*. In some embodiments, the nucleotide sequence encoding the HSD is shown in SEQ ID NO: 11 or 12; the nucleotide sequence encoding the CASDH is shown in SEQ ID NO: 13 or 14; the nucleotide sequence encoding the CASDC is shown in SEQ ID NO: 15; the nucleotide sequence encoding the Linker peptide is shown in SEQ ID NO: 16; the nucleotide sequence encoding the Omp85 is shown in SEQ ID NO: 17, 18 or 19; and the nucleotide sequence encoding the Ag43 is shown in SEQ ID NO: 20.

[0017] In some embodiments, the enzyme protein is a tandem link of multiple enzyme proteins. In some embodiments, the multiple enzyme proteins are the same enzyme protein. To maintain the conformation of the protein or form a polymer, in some embodiments, the enzyme proteins are linked by a linker peptide. The linker peptide is preferably a polypeptide with an amino acid sequence as shown in SEQ ID NO:6.

[0018] In some embodiments, the aforementioned *E. coli* consists of *E. coli* cells displaying HSD, *E. coli* cells displaying CASDH, and *E. coli* cells displaying CASDC. The cell ratio of *E. coli* cells displaying HSD, *E. coli* cells displaying CASDH, and *E. coli* cells displaying CASDC is 1:1:1 to 3:3:2; for example, 1:1:1, 2:1:1, 2:2:1, 1:2:1, 1:2:2, 3:2:1, 3:3:1, or 3:3:2.

[0019] The present invention has the following advantages: This invention constructs an enhanced Ag43 surface display system for whole-cell catalytic synthesis of spermidine. Simultaneously, different linker peptides are used to connect the autotransporter Ag43 and the recombinant protein, reducing steric hindrance and ensuring the normal functioning of the fusion protein domains. This invention also optimizes Ag43 expression at the assembly and transmembrane transport levels, improving the efficiency of bacterial surface display of the recombinant protein. The enzyme selected in this invention can regenerate NAD(P), reducing cofactor requirements. The method for producing spermidine provided by this invention is simple and uses readily available raw materials, showing promising prospects for industrial application. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the catalytic synthesis principle of the present invention; Figure 2 The yield of spermidine in different types of whole-cell catalytic fermentation; Figure 3 It is based on a single promoter multicistronic strategy to construct a BAM complex overexpression system. Detailed Implementation

[0021] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0022] In this invention, the autotransporter protein Ag43 is an endogenous protein of Escherichia coli. It is a typical β-barrel integrated membrane protein, consisting of multiple antiparallel β-sheets forming a transmembrane β-barrel backbone. It is mainly composed of a signal peptide (SP), a passenger domain, an α-helix, and a β-barrel domain. The passenger domain is linked to the β-barrel domain through an α-helix structure. Through the β-barrel formed by the β-barrel domain, the passenger protein is transported across the outer membrane and anchored to the outer membrane of Gram-negative bacteria.

[0023] Example 1: Mutation and Screening of Homoserine Dehydrogenase, Carboxysemine Dehydrogenase, and Carboxysemine Decarboxylase 1. Homoserine dehydrogenase (HSD) Surface display vectors were constructed using a combination of double enzyme digestion and homologous recombination: using schsd-F1 (SEQ ID NO:21) and schsd-R1 (SEQ ID NO:22), according to a 2× Phanta Max Master Mix (P525, Novizan) system and PCR conditions, from *Saccharomyces cerevisiae* (Saccharomyces cerevisiae)... Saccharomyces cerevisiae The homoserine dehydrogenase gene was cloned from the S288C genome. schsd The amino acid sequence was accessed via NCBI with accession number NP_012673.3. The PCR product, as confirmed by 1% agarose gel electrophoresis, was close to its theoretical size of 1077 bp. The vector pET22b-Ag43 was linearized using BamHI and EcoRI via double digestion. The linearized vector and schsd gene fragment were then recovered and purified using an Omerga#D2500 Gel Extraction Kit, following the prescribed procedures. Subsequently, the linearized vector and... schsd Genes were mixed at a 2:1 (v / v) ratio and reacted with homologous recombinase (2 × ClonExpress Mix, Novizan) at 50°C for 15 min to complete the recombination reaction. Following the chemocompetent transformation method, 10 μL of the recombinant product was directly transformed into 50 μL of [a specific enzyme / product]. E. coli DH5α competent cells were cultured on plates containing 100 μg / mL ampicillin resistance to form monoclonal colonies. These monoclonal colonies were then picked, cultured, and sequenced to determine their composition. schsd The gene was successfully inserted between BamHI and EcoRI in the pET22b-Ag43 vector, thus obtaining E. coliDH5α-pET22b-schsd-Ag43 clone strain.

[0024] Construction of the SchHSD surface display expression strain: The seed culture of the cloned strain was re-prepared. 2 mL of the bacterial culture was taken, and the recombinant vector pET22b-schsd-Ag43 was extracted according to the instructions of the plasmid extraction kit (Omega #D6943 Plasmid Mini Kit). This extracted vector was then transformed into a new... E. coli After monoclonalization of BL21 (DE3) competent cells on plates containing 100 μg / mL ampicillin resistance, single colonies were picked for colony PCR. Primers JLPCR-F1 (SEQ ID NO:23) and JLPCR-R1 (SEQ ID NO:24) were used, and colony PCR was performed according to the instructions of 2×Taq PCR MasterMix II (TianGen KT230630). The colony PCR product yielded a 1177 bp band after agarose gel electrophoresis, indicating successful transformation of the recombinant vector and the acquisition of a surface-displaying recombinant expression strain. E. coli BL21-pET22b-schsd-Ag43.

[0025] Construction of schsd gene mutants: To further improve the enzyme's catalytic efficiency and cofactor dependence, a mutant library was constructed using error-prone PCR technology. The specific steps are as follows: Using the pET22b-schsd-Ag43 recombinant plasmid as a template, error-prone DNA templates were prepared by conventional PCR amplification using primers schsd-F2 (SEQ ID NO:25) and schsd-R2 (SEQ ID NO:26) with 2× Phanta Max Master Mix. The DNA fragments (templates) were recovered from the gel and their concentrations were determined. The recovered DNA fragments (templates) were diluted with water to 1 ng / μL, 10 ng / μL, and 100 ng / μL. Error-prone PCR reactions were performed in 30 μL systems as shown in Table 1 (Tianjingsha CAT: 220167), repeated in triplicate, following the procedure in Table 2.

[0026] Table 1 Commonly Misunderstood PCR Reaction Systems Table 2 Commonly Misunderstood PCR Reaction Procedures After the error-prone PCR was completed, 3 μL of each sample was taken for electrophoresis detection.

[0027] Using the yeast shuttle vector pRS425 containing the LEU2 selection marker as the background plasmid, and primers pRS425-schsd-F1 (SEQ ID NO:27) and pRS425-schsd-R1 (SEQ ID NO:28), a linearized vector pRS425-schsd was prepared using novizan high-fidelity enzyme P525 as the background plasmid. This vector, along with a random mutant fragment of SCHSD containing approximately 20 bp homologous arms at both ends of the DNA fragment, was co-transformed into competent *Saccharomyces cerevisiae* cells. Relying on yeast's efficient homologous recombination ability, multi-fragment assembly was completed within the cells, yielding a SCHSD random mutant plasmid library. Positive clones were screened using LEU2 auxotrophic selection plates, and then the library plasmids were extracted using a yeast plasmid extraction kit. schsd Mutant library plasmids.

[0028] To increase plasmid concentration, transformation into *E. coli* DH5α for enrichment was performed: [The following is a list of transformation methods and their corresponding values]. schsd Mutant library plasmids were introduced into *E. coli* BL21(DE3) host to enrich the mutant library and obtain high-quality mutant libraries. schsd Random mutation library.

[0029] Mutant induction culture: All transformants on the plate were picked into 96-well plates containing 200 μL LB liquid medium (100 μg / mL ampicillin and 0.2 mmol / L IPTG) per well and cultured at 37°C and 200 r / min for 5 hours.

[0030] After cultivation, mutant screening was performed: 10 μL of 20 mg / mL snailase was added to a 96-well plate and incubated at 37℃ for 1 h. Then, 0.2 mL of the cell disruption solution was transferred back to the 96-well plate, and 50 μL of 50 mM L-homoserine and 24 μL of 10 mM coenzyme NAD were added respectively, maintaining a total reaction volume of 300 μL, a temperature of 35℃, and a pH of 7.4. The slope of the reduced coenzyme NADH value at 340 nm was measured using an Enspire multi-label assay system microplate reader; a larger slope indicated higher enzyme activity. Furthermore, NAD was replaced with NADP, while maintaining identical catalytic conditions, to induce a coenzyme NADP-dependent mutation. schsd Screening for mutants.

[0031] Finally, SCHSD1, which can efficiently utilize NAD as a specific cofactor, and SCHSD2, which uses NADP as a specific cofactor, were screened with amino acid sequences as shown in SEQ ID NO:1 and SEQ ID NO:2. Their nucleotide sequences are shown in SEQ ID NO:11 and SEQ ID NO:12. The specific enzyme activities of the mutants are 57.8 and 43.2 U / mg, respectively.

[0032] Considering the cost of adding cofactors in spermidine biosynthesis, SCHSD1, which has relatively high catalytic specificity for NAD, was ultimately chosen for the subsequent catalytic reaction.

[0033] Construction of the mutant SchSSD1 surface display vector: The specific method refers to the method for constructing wild-type SchSSD surface display vectors using a combination of double enzyme digestion and homologous recombination. Specifically, the schsd1-F1 (SEQ ID NO:29) and schsd1-R1 (SEQ ID NO:30) primers were used to amplify the mutant schsd1 gene. The methods for double enzyme digestion of other vectors, purification of DNA fragments, homologous recombination ligation of the vector and the inserted gene, and screening and identification of cloning strains remained consistent, thus obtaining… E. coli DH5α- pET22b-schsd1-Ag43 clone strain.

[0034] Preparation of recombinant bacteria displaying the mutant SCHSD1 surface: The specific method follows the construction procedure for SCHSD surface-display expression strains. The surface-display recombinant expression strain was obtained after colony PCR verification. E. coli BL21-pET22b-schsd1-Ag43.

[0035] The specific method for measuring the surface enzyme activity of homoserine dehydrogenase in cells is as follows: The enzyme used is a pre-constructed... E. coli BL21-pET22b-schsd-Ag43 or E. coli BL21-pET22b-schsd1-Ag43 recombinant strain.

[0036] Induction of surface-display enzyme expression: The surface-display recombinant expression strain was transferred at a 4% inoculum to LB medium containing 100 μg / mL ampicillin (5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl). When the cell OD... 600 When the pH reaches 0.8-1.0, add IPTG to a final concentration of 0.2 mM and induce culture at 25°C for 5 h. After induction, collect recombinant cells by centrifugation at 4°C, 5000 rpm, and 5 min. Resuspend cells in phosphate buffer (pH 7.4) to OD. 600 The concentration was 5.0 (cell volume was 5OD), and it was stored at 4°C for later use.

[0037] The scHSD and its mutant surface-display cell catalytic reaction system: 3 mL of reaction volume contained 1 mM L-homoserine, 0.12 mM NAD, and pH 8.0. 5 OD of surface-display cells were added, and the temperature was 35℃. The production rate of the cofactor NADH was measured at 340 nm over 10 min. One unit of enzyme activity (U) is defined as the amount of enzyme required to produce 1 μmol of NADH per min, and specific enzyme activity (U / OD) is defined as the enzyme activity per OD of whole cells. Activity assays showed that the specific activities of wild-type scHSD and the mutant scHSD1 surface-display cells were 23.4 U / OD and 89.4 U / OD, respectively. This indicates a significant increase in enzyme activity after mutation. Therefore, the mutant was used in subsequent experiments to construct a spermidine catalytic synthesis system.

[0038] 2. Carboxysemine dehydrogenase Surface display vectors were constructed using a combination of double enzyme digestion and homologous recombination: pccasdh-F1 (SEQ ID NO:31) and pccasdh-R1 (SEQ ID NO:32) were used to construct the vectors from... Pseudoflavonifractor capillosus The carboxysemine dehydrogenase gene was cloned from the strain. pccasdh Its amino acid sequence accession number on NCBI is UCX62627.1, and the PCR product, after 1% agarose gel electrophoresis, was identified as close to its theoretical size of 1257 bp. Other experiments included linearization of the vector pET22b-Ag43, purification of the DNA fragment, ligation of homologous recombination, and transformation of the ligation product to... E. coli DH5α competent cells were constructed using the same steps as described above for pET22b-schsd-Ag43. After sequencing, the correct recombinant plasmid pET22b-pccasdh-Ag43 was obtained through screening. Preparation of PcCASDH surface-displaying recombinant bacteria: The specific method referred to the construction procedure of the SCHSD surface-displaying expression strain. The universal primers used for colony PCR were the pET22b-Ag43 plasmid primers. The surface-displaying recombinant expression strain was obtained after colony PCR verification. E. coli BL21-pET22b-pccasdh-Ag43.

[0039] Construction of PcCASDH gene mutants: To further improve the enzyme's catalytic efficiency and cofactor dependence, a mutant library was constructed using error-prone PCR technology. The specific procedures were the same as those for constructing the SCHSD gene mutant in section 1, except that the template pET22b-pccasdh-Ag43, mutant primers pccasdh-F2 (SEQ ID NO:33) and pccasdh-R2 (SEQ ID NO:34) were used to prepare an error-prone DNA template. The linearized vector pRS425-pccasdh was prepared as a background plasmid using primers pRS425-pccasdh-F1 (SEQ ID NO:35) and pRS425-pccasdh-R1 (SEQ ID NO:36). The mutant library was then enriched and induced to grow according to the methods described in section 1.

[0040] Mutant screening: 10 μL of 20 mg / mL snailase was added to a 96-well plate and incubated at 37℃ for 1 h. Then, 0.2 mL of the cell disruption solution was transferred back to the 96-well plate, and 5 mM L-aspartic acid β-semialdehyde, 5 mM putrescine, and 2 mM NADH were added to the wells. The total reaction volume was 300 μL, the temperature was 35℃, and the pH was 8.0. The rate of decrease of the reduced coenzyme NADH at 340 nm was detected using an Enspire multilabel assay system. Furthermore, NADH was replaced with NADPH, while maintaining identical catalytic conditions, to screen for NADPH-dependent PcCASDH mutants.

[0041] Finally, PcCASDH1, with amino acid sequences as shown in SEQ ID NO:3 and 4, and PcCASDH2, with NADPH as a specific cofactor, were screened. Their nucleotide sequences are shown in SEQ ID NO:13 and SEQ ID NO:14. The specific enzyme activities of the mutants were 60.6 U / mg and 34.5 U / mg, respectively.

[0042] Considering the cost of adding cofactors in spermidine biosynthesis, PcCASDH1, which has relatively high catalytic specificity for NADH, was selected for subsequent catalytic reactions.

[0043] Construction of the mutant PcCASDH1 surface display vector: The specific method is the same as described in section 1, which uses a combination of double enzyme digestion and homologous recombination to construct the wild-type SCHSD surface display vector. Specifically, pccasdh1-F1 (SEQ ID NO:37) and pccasdh1-R1 (SEQ ID NO:38) were used as primers to amplify the mutant. pccasdh1Genes. The methods for double enzyme digestion of other vectors, purification of DNA fragments, homologous recombination ligation of the vector and the inserted gene, and screening and identification of cloning strains remain consistent, thus obtaining... E. coli DH5α-pET22b-pccasdh1-Ag43 clone strain.

[0044] Preparation of recombinant bacteria displaying the mutant PcCASDH1 surface: The specific method refers to the construction procedure of the ScHSD surface-displaying expression strain described in section 1. After colony PCR verification, the surface-displaying recombinant expression strain was obtained. E. coli BL21-pET22b-pccasdh1-Ag43.

[0045] Following the enzyme-inducible display procedure on the SCHSD surface as described in section 1, E. coli BL21-pET22b-pccasdh-Ag43 and E. coli The recombinant strain BL21-pET22b-pccasdh1-Ag43 was induced to express PcCASDH and its mutant surface display cells.

[0046] The PcCASDH and its mutant surface-display cell catalytic reaction system: 3 mL of reaction volume contained 2 mM L-aspartic acid β-semialdehyde, 2 mM putrescine, 1 mM NADH, pH 8.0, and surface-display cells with a final concentration of 5 OD. The temperature was 35℃, and the rate of NADH reduction was measured at 340 nm. One unit of enzyme activity (U) is defined as the amount of enzyme required to generate or reduce 1 μmol of NADH in 1 min. Specific enzyme activity (U / OD) is defined as the enzyme activity units present in whole cells per OD. Activity assays showed that the specific activities of wild-type PcCASDH and mutant PcCASDH1 surface-display cells were 10.87 U / OD and 25.78 U / OD, respectively. This indicates a significant increase in enzyme activity after mutation. Therefore, the mutant was used in subsequent experiments to construct a spermidine catalytic synthesis system.

[0047] 3. Carboxysemine decarboxylase Surface display vectors were constructed using a combination of double enzyme digestion and homologous recombination: cjcasdc-F1 (SEQ ID NO:39) and cjcasdc-R1 (SEQ ID NO:40) were used to construct the vectors from... Campylobacter jejuni The carboxysylspermine decarboxylase gene was cloned from subsp. jejuni 81116. cjcasdcIts amino acid sequence accession number (UUR11295.1) on NCBI is [not specified]. 1% agarose gel electrophoresis confirmed its size to be close to the theoretical value of 1149 bp. Other experiments included linearization of the vector pET22b-Ag43, purification of the DNA fragment, ligation of homologous recombination, and transformation of the ligation product to [not specified]. E. coli The construction steps for DH5α competent cells were completely consistent with those for pET22b-schsd-Ag43. Finally, after sequencing by Shanghai Sangon Biotech, the correct recombinant plasmid pET22b-cjcasdc-Ag43 was obtained.

[0048] Preparation of CjCASDC surface-displaying recombinant bacteria: The specific method refers to the construction procedure of SCHSD surface-displaying expression strains. After colony PCR verification, the surface-displaying recombinant expression strains were obtained. E. coli BL21-pET22b-cjcasdc-Ag43.

[0049] Construction of CjCASDC gene mutants: To further improve the enzyme's catalytic efficiency, a mutant library was constructed using error-prone PCR technology. The specific procedures were the same as those for constructing the SCHSD gene mutant in section 1, except that the template pET22b-cjcasdc-Ag43, mutant primers cjcasdc-F2 (SEQ ID NO:41) and cjcasdc-R2 (SEQ ID NO:42) were used to prepare an error-prone DNA template. The linearized vector pRS425-cjcasdc was prepared as a background plasmid using primers pRS425-cjcasdc-F1 (SEQ ID NO:43) and pRS425-cjcasdc-R1 (SEQ ID NO:44). The mutant library was then enriched and induced to grow according to the methods described in section 1.

[0050] Screening of the CjCASDC mutant library: 10 μL of 20 mg / mL snailase was added to a 96-well plate and incubated at 37℃ for 1 h. Then, 0.2 mL of the cell disruption solution was transferred back to the 96-well plate, and 2 mM carboxysemine, 0.5 mM dithiothreitol (DTT), and 0.1 mM pyridoxal phosphate (PLP) were added to a final volume of 0.3 mL. The pH was adjusted to 8.0, and the plate was incubated at 35℃ for 30 min to catalyze the reaction and obtain the reaction solution.

[0051] The content of spermidine in the reaction solution was determined by high performance liquid chromatography (HPLC), and the activity of different CjCASDC mutants was calculated based on the spermidine content. The specific operation was performed according to the method described in GB 5009.208-2016 National Food Safety Standard, Determination of Biogenic Amines in Food: The reaction solution was taken, centrifuged, and 0.3 mL of the supernatant was collected. 1 mL of saturated sodium bicarbonate solution, 100 μL of sodium hydroxide solution (1 M), and 1 mL of derivatization reagent (10 mg / mL dansyl chloride in acetone solution) were added sequentially. After vortexing for 1 min, the mixture was placed in a 60℃ constant temperature water bath for 15 min for derivatization. Then, 100 μL of sodium glutamate solution was added separately, and the mixture was shaken and reacted at 60℃ for 15 min. Remove the samples and cool to room temperature. Add 1 mL of water to each centrifuge tube, vortex for 1 min, and remove approximately 1 mL of acetone by nitrogen purging under a 40°C water bath. Add 0.5 g of sodium chloride and vortex until completely dissolved. Add 5 mL of diethyl ether and vortex for 2 min. After standing and separating the layers, aspirate the upper organic phase (ether layer) and extract again. Combine the ether extracts and dry under nitrogen purging under a 40°C water bath. Add 1 mL of acetonitrile and vortex to completely dissolve the residue. Filter the solution through a 0.22 μm syringe filter into a vial for analysis.

[0052] Reference conditions for liquid chromatography: C18 column (250 mm length, 4.6 mm inner diameter, 5 μm packing particle size), UV detection wavelength 254 nm, injection volume 20 μL, column temperature 35℃, mobile phase A: 90% acetonitrile / 10% 0.01 mol / L ammonium acetate solution (containing 0.1% acetic acid) (v / v), mobile phase B: 10% acetonitrile / 90% 0.01 mol / L ammonium acetate solution (containing 0.1% acetic acid) (v / v), flow rate 0.8 mL / min. Gradient elution program is shown in Table 3.

[0053] Table 3 Gradient elution program Finally, a mutant CjCASDC1, with an amino acid sequence as shown in SEQ ID NO:5, was screened and found to be capable of efficiently catalyzing the synthesis of spermidine. Its nucleotide sequence is shown in SEQ ID NO:15. The specific enzyme activity of the mutant was 89.5 U / mg, while the activity of the wild-type CjCASDC was 38.7 U / mg. The mutant activity was 2.3 times higher than that of the wild-type. Therefore, the mutant CjCASDC1 was used as the research object in subsequent experiments.

[0054] Construction of the surface display vector for the mutant CjCASDC1: The specific method referred to the method for constructing the wild-type SCHSD surface display vector using a combination of double enzyme digestion and homologous recombination. Specifically, the mutant cjcasdc1 gene was amplified using primers cjcasdc1-F1 (SEQ ID NO:45) and cjcasdc1-R1 (SEQ ID NO:46). The methods for double enzyme digestion of other vectors, purification of DNA fragments, homologous recombination ligation of the vector and the inserted gene, and screening and identification of cloned strains remained consistent, thus obtaining… E. coli DH5α- pET22b-cjcasdc1-Ag43 clone strain.

[0055] Preparation of surface-displaying recombinant bacteria from mutant CjCASDC1: The specific method referred to the construction procedure of surface-displaying expression strains in SCHSD. The surface-displaying recombinant expression strain was obtained after colony PCR verification. E. coli BL21-pET22b-cjcasdc1-Ag43.

[0056] Following the procedure for enzyme induction on the SCHSD surface, E. coli BL21-pET22b-cjcasdc-Ag43 or mutant E. coli The recombinant strain BL21-pET22b-cjcasdc1-Ag43 was induced to express CjCASDC and its mutant CjCASDC1 surface display cells.

[0057] Assay for the activity of surface-displayed cells containing carboxysemine: A 3 mL reaction system containing 5 mM carboxysemine, 50 mM phosphate buffer (pH 8.0), 0.5 mM DTT, and 0.1 mM PLP was added. 5 OD of surface-displayed cells were added, and the reaction was incubated at 35°C for 30 min. The reaction was then terminated by adding 1 mL of 10% trichloroacetic acid. After centrifugation at 8000 rpm for 5 min, the amount of spermine produced was analyzed by HPLC. Specific enzyme activity (U) is expressed as the amount of spermine produced per OD of whole cells per minute. The specific activities of wild-type CjCASDC and mutant CjCASDC1 surface-displayed cells were 32.56 U / OD and 58.72 U / OD, respectively. This shows that the mutant enzyme activity was significantly increased. Therefore, the mutant was used in subsequent experiments to construct a spermine-catalyzed synthesis system.

[0058] Example 2: Co-expression and mono-expression of surface-display cells for catalytic synthesis of spermidine according to Figure 1 The process shown constructs a surface display system for the catalytic synthesis of spermidine.

[0059] 1. Simultaneous expression of three enzymes to construct surface display cells Using the constructed pETDuet-schsd1-Ag43 vector as a template, the vector was amplified using primers pet22b-3schsd1-F / R to obtain the linearized plasmid pet22b-3schsd1. Using the constructed pETDuet-pccasdh1-Ag43 and pETDuet-cjcasdc1-Ag43 as templates, and primers 3pccasdh1-F / R and 3cjcasdc1-F / R as templates, fragments 3pccasdh1 and 3cjcasdc1 were obtained. Following the procedure described in Example 1, the linearized plasmid and PCR product fragments were purified using a gel extraction kit. A 10 μL recombinant reaction mixture was prepared on ice: 2 μL linearized plasmid, 1 μL 3pccasdh1 fragment, 1 μL 3cjcasdc1 fragment, 5 μL 2×ClonExpress Mix, and 1 μL ddH2O. After thorough mixing, the mixture was incubated at 50 °C for 25 min, then immediately cooled on ice. Subsequently, the 10 μL ligation product was converted to [the desired product] using a chemicompetent cell conversion method. E. coli DH5α competent cells were screened for single colonies using ampicillin-resistant plates. Following the method described in Example 1, colony PCR was performed using JLPCR-F1 (SEQ ID NO:23) / R1 (SEQ ID NO:24), yielding PCR products around 3858 bp. The corresponding single colonies were sent to Shanghai Sangon Biotech for sequencing to ensure successful plasmid ligation. Finally, recombinant strains were screened. E. coli DH5α-pET22b-schsd1-pccasdh1-cjcasdc1-Ag43.

[0060] Table 4 Primer Sequences Using a plasmid extraction kit, the recombinant plasmid pET22b-schsd1-pccasdh1-cjcasdc1-Ag43 was extracted and transformed into a new cell using the chemically competent transformation method. E. coli BL21 (DE3) competent cells, after verification by JLPCR-F1 / R1, yielded a correctly transformed recombinant expression strain. E. coli BL21-pET22b-schsd1-pccasdh1-cjcasdc1-Ag43. Cells displayed on the surface were induced according to the method described in Example 1.

[0061] 2. Expressing three types of surface display cells respectively. The strains used were the three surface display cells constructed in Example 1, namely... E. coli BL21-pET22b-schsd1-Ag43,E. coli BL21-pET22b-pccasdh1-Ag43 and E. coli BL21-pET22b-cjcasdc1-Ag43. Cells displayed on the surface were induced according to the method described in Example 1.

[0062] 3. Constructing a surface-displaying whole-cell catalytic system A whole-cell catalytic system co-expressing three enzymes was established on the surface: the surface-displaying cells had a wet weight of 200 g / L, L-homoserine 20 g / L, putrescine 20 g / L, NAD 5 g / L, ATP 1 g / L, DTT 0.1 g / L, and PLP 1 g / L. The reaction was carried out at 35°C for 24 hours. A 1 mL sample was taken, and spermidine was quantitatively analyzed according to the method described in Example 1.

[0063] Three enzymes were separately displayed to construct a whole-cell catalytic system: the surface-displayed cells had a wet weight of 200 g / L (different surface-displayed cells were mixed in a 1:1:1 wet weight ratio), L-homoserine 20 g / L, putrescine 20 g / L, NAD 5 g / L, ATP 1 g / L, DTT 0.1 g / L, and PLP 1 g / L; the reaction was carried out at 35°C for 24 hours. A 1 mL sample was taken, and spermidine was quantitatively analyzed according to the method described in Example 1.

[0064] Whole-cell catalysis revealed that the three surface-displaying cell types were pET22b. - The combination of schsd1-Ag43, pET22b-afcasdh1-Ag43, and pET22b-cjcasdc1-Ag43 produced the highest amount of spermidine, reaching 2.66 g / L, while the catalytic efficiency of the system co-expressing all three enzymes was lower (1.31 g / L). This suggests that displaying a single enzyme introduces less survival pressure on cell growth and may be more conducive to enzyme display and activity. Therefore, subsequent whole-cell catalytic experiments were conducted using cells displaying a single enzyme.

[0065] Example 3: Linker peptide-optimized surface-displaying cells for catalytic synthesis of spermidine 1. Optimization of Linker Peptides with Different Properties Four different linker peptides with nucleotide sequences as shown in SEQ ID NO:53-56 were selected. Codon optimization was performed based on E. coli preferences, and primers were designed directly. The specific experimental procedure was as follows: pET22b-schsd1-Ag43, pET22b-pccasdh1-Ag43, and pET22b-cjcasdc1-Ag43 were used as templates, and pet22b-FF / pet22b-R, pet22b-RF / pet22b-R, pet22b-FR-F / pet22b-R, and pet22b-F-RF / pet22b-R were used as primers. Linearized plasmids pet22b-F, pet22b-R, pet22b-FR, and pet22b-RF were obtained by primer amplification; fragments schsd1-F1 / schsd1-FR, schsd1-FR, and schsd1-RF were obtained by amplification using primers schsd1-F1 / schsd1-FR, schsd1-F1 / schsd1-RR, schsd1-F1 / schsd1-FR-R, and schsd1-F1 / schsd1-RF-R; and the ligation products were transformed according to the novitol-derived recombinase ligation system in Example 1. E. coliThe DH5α competent cells were used to select single colonies for culturing in liquid and then sent to Shanghai Sangon Biotech for sequencing to screen for successfully constructed recombinant plasmids pet22b-schsd1-F-Ag43, pet22b-schsd1-R-Ag43, pet22b-schsd1-FR-Ag43, and pet22b-schsd1-RF-Ag43. Similarly, using primers pccasdh1-F1 / pccasdh1-FR, pccasdh1-F1 / pccasdh1-RR, pccasdh1-F1 / pccasdh1-FR-R, pccasdh1-F1 / pccasdh1-RF-R, cjcasdc1-F1 / cjcasdc1-FR, cjcasdc1-F1 / cjcasdc1-RR, cjcasdc1-F1 / cjcasdc1-FR-R, and cjcasdc1-F1 / cjcasdc1-RF-R, fragments pccasdh1-F, pccasdh1-R, pccasdh1-FR, pccasdh1-RF, cjcasdc1-F, cjcasdc1-R, cjcasdc1-FR, and cjcasdc1-RF were amplified and ligated into the linearized vectors described above, thereby constructing the recombinant plasmid pet22b- pccasdh1-F-Ag43, pet22b- pccasdh1-R-Ag43, pet22b- pccasdh1-FR-Ag43, pet22b- pccasdh1-RF-Ag43, pet22b- cjcasdc1-F-Ag43, pet22b- cjcasdc1-R-Ag43, pet22b- cjcasdc1-FR-Ag43, and pet22b- cjcasdc1-RF-Ag43. Plasmids were extracted using a plasmid extraction kit from... E. coli The plasmids described above were extracted from the DH5α cloning host and transformed into expression hosts, respectively. E. coli In BL21 (DE3) competent cells, positive recombinants were screened using ampicillin-resistant plates, and colony PCR was performed using JLPCR-F / R universal primers to verify successful plasmid transformation. Subsequently, different surface-displaying recombinant expression strains were constructed.

[0066] Table 5 Design of Linker peptide primers Whole-cell catalytic synthesis of spermidine was performed using the single enzyme surface display method described in Example 2.

[0067] Table 6. Results of different strains and their catalytic synthesis of spermidine Analysis of the whole-cell catalytic synthesis of spermidine yield results showed that the three surface-displaying cell types were respectively E. coli pET22b-schsd1-F-Ag43 E. coli / pET22b-afcasdh1-F-Ag43 and E. coli The combination mode of / pET22b-cjcasdc1-F-Ag43 produced the highest amount of spermidine, reaching 4.78 g / L, further indicating that flexible linker peptides are more conducive to the functional display of enzyme proteins.

[0068] 2. Flexible Linker Peptides of Different Lengths The extended linker peptide F9 (GGGGG)9, with an amino acid sequence as shown in SEQ ID NO:6 and a nucleotide coding sequence as shown in SEQ ID NO:16, was designed and sent to Shanghai Sangon Biotech Co., Ltd. for nucleotide sequence synthesis. Using the synthesized F9 as a template, the F9 fragment was amplified using F9-F / R primers. Using pET22b-schsd1-Ag43, pET22b-pccasdh1-Ag43, and pET22b-cjcasdc1-Ag43 as templates, and primers Ag43-F9-F / schsd1-F9-R, Ag43-F9-F / pccasdh1-F9-R, and Ag43-F9-F / cjcasdc1-F9-R, the fragments pET22b-schsd1-F9, pET22b-pccasdh1-F9, and pET22b-cjcasdc1-F9 were amplified to obtain the same sequences. Following the homologous recombination method described in Example 1, recombinant plasmids pET22b-schsd1-F9-Ag43, pET22b-pccasdh1-F9, and pET22b-cjcasdc1-F9-Ag43 were constructed. These plasmids were then transformed into expression hosts. E. coli BL21 (DE3) competent cells were used to screen for successfully transformed recombinant expression strains. E. coli BL21 / pET22b-schsd1-F9-Ag43, E. coli BL21 / pET22b-pccasdh1-F9 and E. coli BL21 / pET22b-cjcasdc1-F9-Ag43. Surface-displaying cells were obtained following the induction expression steps for surface-displaying cells in Example 1.

[0069] Table 7 Primer Sequences Following the single-enzyme surface-display whole-cell catalytic system described in Example 2, the efficiency of spermidine synthesis in the surface-display system was assessed. Compared to the flexible short linker peptide (F), extending the linker peptide (F9) increased the spermidine synthesis yield, reaching 5.38 g / L.

[0070] Example 4: Dual-copy gene co-displays whole-cell use for catalytic synthesis of spermidine The recombinant plasmids pET22b-schsd1-F9-Ag43, pET22b-pccasdh1-F9, and pET22b-cjcasdc1-F9-Ag43 were digested with the restriction endonuclease EcoRI to obtain the linearized vectors pET22b-schsd1-F9-Ag43, pET22b-pccasdh1-F9, and pET22b-cjcasdc1-F9-Ag43. Using recombinant plasmids pET22b-schsd1-F9-Ag43, pET22b-pccasdh1-F9, and pET22b-cjcasdc1-F9-Ag43 as templates, the target fragments schsd1-F9, pccasdh1-F9, and cjcasdc1-F9 were amplified using primers schsd1-F1 / schsd1-F9-R, pccasdh1-F1 / pccasdh1-F9-R, and cjcasdc1-F1 / cjcasdc1-F9-R. After purifying the linearized vectors and PCR products, the corresponding fragments were ligated to the linearized vectors using homologous recombination and introduced into the target vectors. E. coli After DH5α competent cells were cultured, single colonies were picked from ampicillin-containing antibiotic plates, cultured in liquid, and then sent to Shanghai Sangon Biotech for sequencing to screen for correctly ligated recombinant plasmids pET22b-schsd1-F9-schsd1-F9-Ag43, pET22b-pccasdh1-F9-pccasdh1-F9-Ag43, and pET22b-cjcasdc1-F9-cjcasdc1-F9-Ag43. After successful sequencing, the cells were processed according to Example 1. E. coli The BL21 (DE3) competent cell transformation method involves transforming the above recombinant plasmid into... E. coli BL21 (DE3) competent cells were used to construct a double-copy gene co-expression strain. E. coli / pET22b-schsd1-F9-schsd1-F9-Ag43、 E. coli / pET22b-pccasdh1-F9-pccasdh1-F9-Ag43 and E. coli / pET22b-cjcasdc1-F9-cjcasdc1-F9-Ag43.

[0071] Following the induced expression procedure described in Example 2, whole cells co-displayed with three dual-copy genes were obtained. Spermine biocatalytic synthesis was then performed at a whole-cell catalytic ratio of 1:1:1, and the efficiency of spermine synthesis in the surface display system was tested. The results showed that by constructing surface display cells expressing dual-copy genes, the yield of spermine synthesized reached 6.38 g / L. This indicates that after anchoring, dual-copy genes more easily form dimers on the cell surface, exhibiting relatively high enzyme catalytic activity.

[0072] Example 5: Effect of assembled proteins on bacterial surface display efficiency 1. Overexpression of Omp85 assembly protein enhances bacterial surface display efficiency. Cloning originates from Thermus thermophilus The gene encoding the Omp85 protein in HB27 ttomp85 The amino acid sequence of the gene is the sequence with accession NO. AAS80541.1 on NCBI. This protein-coding gene was ligated into the BamHI / EcoRI space of the plasmid pRSFDuet-1 to construct the recombinant expression plasmid pRSFDuet-ttomp85. Specifically, the pRSFDuet-1 vector was digested with BamHI and EcoRI to obtain the linearized vector pRSFDuet. Primers ttomp85-F1 (SEQ ID NO:80) and ttomp85-R1 (SEQ ID NO:81) were used to express the gene. T. thermophilus Using the HB27 genome as a template, the target gene was amplified. ttomp85 The fragment, identified by 1% agarose gel electrophoresis as being consistent with its theoretical size of 2493 bp, was purified using a gel extraction kit using the linearized vector and PCR product. It was then ligated according to the homologous recombination method described in Example 1 and transformed. E. coli After DH5α competent cells were collected, they were screened using kanamycin-containing (50 μg / mL) antibiotic plates. Single colonies were picked and cultured in liquid culture for colony PCR verification, followed by sequencing at Shanghai Sangon Biotech. This process identified the correctly ligated recombinant plasmid pRSFDuet-ttomp85. Further, this plasmid was transformed into expression hosts. E. coli BL21 (DE3) competent cells were used to select single colonies on plates for colony PCR using universal primers T7 (SEQ ID NO:82) and T7t (SEQ ID NO:83) to screen for successfully transformed expression hosts. E. coliBL21 / pRSFDuet-ttomp85. Following the protein induction expression method described in Example 1, the TtOmp85 protein (97.5 kDa) was obtained by 8% SDS-PAGE verification, showing a size close to the theoretical value, indicating that this protein can be expressed... E. coli Successfully expressed in BL21 (DE3).

[0073] Based on plasmid co-expression, the pRSFDuet-ttomp85 plasmid was co-expressed with the plasmid in Example 4 in the expression host. The specific operation process is as follows: preparation E. coli / pET22b-schsd1-F9-schsd1-F9-Ag43、 E. coli / pET22b-pccasdh1-F9-pccasdh1-F9-Ag43 and E. coli Seed culture of / pET22b-cjcasdc1-F9-cjcasdc1-F9-Ag43 was inoculated at 1% onto fresh LB medium (containing 100 μg / mL ampicillin resistance) and cultured at 37°C and 200 rpm until OD. 600 After reaching a concentration of 0.8, the bacterial cells were collected by centrifugation at 5000 rpm for 5 min at 4°C. The bacterial cells were then resuspended in 20 mL of 0.1 M CaCl2 and centrifuged at 5000 rpm for 5 min at 4°C to enrich the cells. This resuspension and enrichment process was repeated once. After resuspending the cells in 1 mL of 0.1 M CaCl2, the solution was aliquoted into 100 μL containers to prepare the final product. E. coli / pET22b-schsd1-F9-schsd1-F9-Ag43、 E. coli / pET22b-pccasdh1-F9-pccasdh1-F9-Ag43 and E. coli / pET22b-cjcasdc1-F9-cjcasdc1-F9-Ag43 competent cells were stored at -80°C. Subsequently, they were processed according to the method described in Example 1. E. coli The BL21 (DE3) competent cell transformation method involved transforming the pRSFDuet-ttomp85 plasmid into the prepared competent cells for screening. Unlike Example 1, the plates used were those containing 25 μg / mL kanamycin and 50 μg / mL ampicillin. Single colonies that grew were considered to be strains containing both plasmids, and colony PCR verification was performed using universal primers T7 / T7t and JLPCR-F1 / R1, respectively. Finally, enhanced surface display cells were constructed. E. coli / pET22b-schsd1-F9-schsd1-F9-Ag43+pRSFDuet-ttomp85 , E. coli / pET22b-pccasdh1-F9-pccasdh1-F9-Ag43 + pRSFDuet-ttomp85 and E. coli / pET22b-cjcasdc1-F9-cjcasdc1-F9-Ag43 + pRSFDuet - ttomp85, thereby improving the assembly and insertion efficiency of Ag43. The above-mentioned bacteria were induced to express the whole-cell catalyst according to the steps of Example 1. The efficiency of spermidine synthesis in the surface display system was tested according to the whole-cell transformation conditions of Example 2.

[0074] Based on whole-cell catalysis results, it was found that the enhanced surface display system can improve whole-cell catalytic efficiency, with spermidine yield reaching 12.08 g / L. Omp85 family proteins are mainly responsible for the recognition, folding, insertion, and assembly of β-barrel outer membrane proteins. Therefore, theoretically, improving the assembly ability of Omp85, such as BamA or heterologous Omp85, could increase Ag43 insertion efficiency, passenger protein display amount, display efficiency of large fusion proteins, and multi-enzyme cascade surface catalytic efficiency.

[0075] 2. Overexpression E. coli BAM system enhances bacterial surface display efficiency Will come from E. coli The BAM complex encoding gene of str. K-12 substr. MG1655 ecbamA, ecbamB, ecbamC, ecbamD, ecbamE The single-promoter, multi-cis-transfer co-expression scheme was used to ligate the pRSFDuet-1 vector ( Figure 3 The specific method is as follows: pRSFDuet-1 was obtained by double digestion of the linearized vector with EcoRI / BamHI. The fragment was then amplified using primers BamA-F / R, BamB-F / R, BamC-F / R, BamD-F / R, and BamE-F / R (Table 8). ecbamA, ecbamB, ecbamC, ecbamD, ecbamE Fragments. The difference in PCR conditions is that... ecbamA The extension time is set to 2 minutes. ecbamB, ecbamC, ecbamD and ecbamE The extension time was set to 1 min. After PCR, the corresponding bands were confirmed by 1% agarose gel electrophoresis to obtain fragments of the theoretical size. ecbamA It is 2433 bp. ecbamB It is 1179 bp. ecbamC, It is 1035 bp. ecbamD It is 738 bp.ecbamE The length is 342 bp. Each gene was amplified using primers, and each gene contained RBS binding sites (RBS1, RBS2, RBS3, and RBS4, Table 8). The linearized vector was mixed. ecbamA, ecbamB, ecbamC, ecbamD and ecbamE Five PCR products were prepared into 20 μL solutions according to the homologous recombination reagent instructions and incubated at 50°C for 60 min. The assembled products were then transformed into... E. coli DH5α competent cells were plated on LB agar plates containing 50 μg / mL kanamycin. Twelve single colonies were picked and transferred to LB liquid containing the same concentration of antibiotic for 12 h. 0.5 mL of the bacterial culture was then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The correctly ligated recombinant plasmid pRSFDuet-ecbamA-ecbamB-ecbamC-ecbamD-ecbamE was obtained. Figure 3 ).

[0076] Table 8 Primers and RBS sequences used The recombinant plasmid pRSFDuet-ecbamA-ecbamB-ecbamC-ecbamD-ecbamE was extracted and transformed into the expression host cell according to the method described in Example 1. E. coli BL21 (DE3) competent cells were used, but single colony selection was performed on LB agar plates containing 50 μg / mL kanamycin. A small-scale expression assay was first performed to verify successful transformation and expression of the recombinant plasmid. Recombinant strain expression was induced according to the method described in Example 1, except that the IPTG concentration was 0.2 mM, the temperature was 16°C, and the rotation speed was 100 rpm. After 12 h, protein expression was detected using 12% SDS-PAGE, and five proteins were successfully expressed.

[0077] The recombinant plasmid pRSFDuet-ecbamA-ecbamB-ecbamC-ecbamD-ecbamE was extracted and transformed into [various molecules]. E. coli / pET22b-schsd1-F9-schsd1-F9-Ag43、 E. coli / pET22b-pccasdh1-F9-pccasdh1-F9-Ag43 and E. coliIn the / pET22b-cjcasdc1-F9-cjcasdc1-F9-Ag43 competent cells, screening was performed using double-antibiotic plates as described in Example 1. The resulting single colonies were identified as strains containing two plasmids. Three single colonies were randomly selected from the double-antibiotic plates and transferred to LB broth of the same concentration for cultivation to obtain a seed culture. Recombinant bacterial expression was induced as described in Example 1, except that the IPTG concentration was 0.2 mM, the temperature was 16°C, and the rotation speed was 100 rpm. After 12 h, protein expression was detected using 12% SDS-PAGE. The expression of five proteins and the surface-displayed protein was detected by 12% SDS-PAGE, and the expression of each protein was successfully achieved.

[0078] The recombinant E. coli that successfully expressed the above protein was re-induced to express the whole cell catalyst, and the efficiency of spermidine synthesis in the surface display system was tested according to the whole cell transformation conditions in Example 2.

[0079] Based on whole-cell catalysis results, it was found that the BAM complex-enhanced surface display system improved whole-cell catalytic efficiency, achieving a spermidine yield of 9.65 g / L. However, the spermidine synthesis efficiency was lower than that of the TtOmp85-enhanced system. This indicates that, compared to the multi-subunit BAM complex, TtOmp85 protein alone can enhance the efficiency of surface display proteins and whole-cell catalysts without the assistance of other proteins. Furthermore, expressing TtOmp85 protein places less pressure on the cells to overexpress exogenous proteins.

[0080] 3. TtOmp85 sequence optimization to enhance bacterial surface display efficiency ttomp85 Construction of gene mutants: To further improve the efficiency of TtOmp85 assembly of outer membrane proteins, a mutant library was constructed using error-prone PCR technology according to the method described in Example 1. The difference was that the pRSFDuet-ttomp85 recombinant plasmid was used as a template, and error-prone PCR primers ttomp85-F2 (SEQ ID NO:98) and ttomp85-R2 (SEQ ID NO:99) were used to obtain an error-prone DNA template and a mutant library. A linearized vector pRS425 was prepared using primers pRS425-ttomp85-F1 (SEQ ID NO:100) and pRS425-ttomp85-R1 (SEQ ID NO:101), and both were transformed into competent Saccharomyces cerevisiae cells to obtain... ttomp85 Random mutant plasmid library. Continuing with the method described in section 1, positive clones were screened using the LEU2 auxotrophic selection plate, and the library plasmids were extracted using a yeast plasmid extraction kit. ttomp85 Mutant library plasmids.

[0081] ttomp85 Enrichment of mutant libraries: High-quality mutant libraries were obtained according to the method described in Example 1. ttomp85 The difference is that the random mutation library is transformed into a pre-preserved laboratory mutant library. E. coli -pet22b-EGFP-Ag43 competent cells were used to display green fluorescent protein (EGFP) (GenBank: OQ870305.1) on the cell surface via the Ag43 autotransporter. Changes in fluorescence intensity allowed for screening of cells utilizing TtOmp85 to significantly enhance the Ag43 autotransporter and its efficiency in displaying the exogenous protein EGFP. Furthermore, single colonies were screened using double-antibiotic plates (25 μg / mL kanamycin and 50 μg / mL ampicillin) to obtain... ttomp85 Random mutant.

[0082] ttomp85 Induction, culture, and screening of random mutants: The procedures were the same as in Example 1, except that liquid culture was performed using dual antibiotics (25 μg / mL kanamycin and 50 μg / mL ampicillin), and wild-type mutants were included. Tt Omp85 was the positive control, the empty vector (pET22b-EGFP-Ag43+pRS425) was the negative control, and the one containing only culture medium was the blank control.

[0083] Screening of TtOmp85 mutants: OD values ​​of each well were measured using a microplate reader. 600 As the cell growth rate; then the EGFP fluorescence intensity was detected to obtain the relative fluorescence units (RFU) for each blank; the OD was adjusted by subtracting the influence of the blank culture medium background. 600 After normalization, the relative EGFP fluorescence intensity S of the mutant was calculated using the following formula: , Where Mut: mutant, WT: wild type, RFU Mut Fluorescence intensity of TtOmp85 mutant wells, RFU Blank Fluorescence background value of blank wells, OD Mut OD of TtOmp85 mutant culture medium 600 Value, OD Blank OD of blank culture medium 600 Value. Normalize WT to 1.00, and for 96-well plate screening, select mutants with S>1.5.

[0084] By screening for mutants with strong fluorescence intensity, TtOmp85 mutants that significantly improve the display efficiency of exogenous proteins were obtained. After sequencing, three mutants were selected—TtOmp85-1, TtOmp85-2, and TtOmp85-3—with relative fluorescence intensities of 1.78, 1.94, and 2.37, respectively. Their amino acid sequences are shown in SEQ ID NO:7-9, and their corresponding nucleotide sequences are shown in SEQ ID NO:16-18.

[0085] Construction of co-expression strains: Plasmids of the three mutants were extracted. Using primers ttomp85-F1 / R1, mutant fragments ttomp85-1, ttomp85-2, and ttomp85-3 were amplified using the three mutant plasmids as templates. Following the homologous recombination and single-colony screening methods described in section 1, these fragments were ligated into the linearized vector pRSFDuet-1 to construct the mutant expression vectors pRSFDuet-ttomp85-1, pRSFDuet-ttomp85-2, and pRSFDuet-ttomp85-3. Subsequently, based on plasmid co-expression, the pRSFDuet-ttomp85 plasmid was co-expressed with the plasmid from Example 2 in the expression host according to the method described in section 1 to obtain surface-display strains with different enhancements. E. coli / pET22b-schsd1-F9-schsd1--Ag43+pRSFDuet-ttomp85-1 , E. coli / pET22b-pccasdh1-F9-pccasdh1-Ag43 + pRSFDuet-ttomp85-1 and E. coli / pET22b-cjcasdc1-F9-cjcasdc1-Ag43 + pRSFDuet - ttomp85-1, E. coli / pET22b-schsd1-F9-schsd1--Ag43+pRSFDuet-ttomp85-2 , E. coli / pET22b-pccasdh1-F9-pccasdh1-Ag43 + pRSFDuet-ttomp85-2 and E. coli / pET22b-cjcasdc1-F9-cjcasdc1-Ag43 + pRSFDuet - ttomp85-2, E. coli / pET22b-schsd1-F9-schsd1--Ag43+pRSFDuet-ttomp85-3, E. coli / pET22b-pccasdh1-F9-pccasdh1-Ag43 + pRSFDuet-ttomp85-3 and E. coli / pET22b-cjcasdc1-F9-cjcasdc1-Ag43 + pRSFDuet - ttomp85-3. The efficiency of spermidine synthesis in the surface display system was tested according to the whole-cell transformation conditions of Example 2. The whole-cell catalysis results are shown in Table 9.

[0086] Table 9 Results of mutants enhancing whole-cell catalysis Based on whole-cell catalysis results, it was found that compared with the wild-type TtOmp85 assembly protein, different TtOmp85 mutants could improve Ag43-mediated enzyme surface display to varying degrees, and further enhance the whole-cell catalytic synthesis of spermidine. However, higher assembly activity or surface display ability of TtOmp85 mutants is not necessarily more beneficial to the synthesis of the final product. Although some mutants showed higher TtOmp85-related activities in the early fluorescence screening or surface display evaluation, their corresponding spermidine yields were not further improved. For example, in this study, the TtOmp85-2 system obtained the highest spermidine yield of 15.63 g / L, while the spermidine yield of another candidate mutant with higher activity was only 14.75 g / L, indicating that there is a significant non-linear relationship between TtOmp85 activity and final catalytic performance.

[0087] This phenomenon may be related to the overall balance of the multi-enzyme surface display system. The main function of TtOmp85 is to promote the insertion and assembly of Ag43 β-barrel structures in the outer membrane. Therefore, increasing TtOmp85 activity within a certain range is beneficial for increasing the surface display level of fusion enzymes such as SCHSD, CASDH, and CASDC. However, when TtOmp85 activity is further enhanced, excessive outer membrane protein assembly may increase the burden on cell membrane protein folding and outer membrane homeostasis, leading to membrane structure perturbation, restricted cell growth, or redistribution of metabolic resources. At this point, even if the surface display level per unit cell is further increased, it may not necessarily translate into higher overall catalytic capacity. Simultaneously, the final spermidine yield is also affected by multiple factors, including cofactor supply, substrate mass transfer, cell viability, and membrane surface space constraints. Once surface display reaches a certain level, the main limiting factor of the system may shift from membrane protein assembly efficiency to enzyme catalytic capacity, cofactor regeneration, or substrate supply. In this case, the marginal benefit of further increasing TtOmp85 activity will significantly decrease, and may even reduce the final catalytic performance due to increased cellular burden.

[0088] Example 6: Optimization of conditions for surface-displaying cells to catalyze spermidine synthesis 1. Different cell ratios The optimal bacterial surface display system constructed in Example 5 was selected: E. coli / pET22b-schsd1-F9-schsd1-Ag43+pRSFDuet-ttomp85-2 (cell A). E. coli / pET22b-pccasdh1- F9-pccasdh1-Ag43 + pRSFDuet-ttomp85-2 (cell B) and E. coli / pET22b-cjcasdc1-F9-cjcasdc1-Ag43 + pRSFDuet - ttomp85-2 (cell C) was used to optimize the proportion of whole-cell catalyst types and catalyze according to the whole-cell catalysis conditions in Example 2. The proportion of display protein used was controlled by regulating the protein usage using 8% SDS-PAGE. Specifically, surface-display cells after induced expression were collected, resuspended in 1× Protein Loading buffer, and cell OD was controlled. 600 The concentration was 5, or 5 OD. After boiling at 98°C for 5 min, centrifuged at 12000 rpm for 1 min, and a 5 μL sample was taken as the loading volume. The protein samples were electrophoresed at 80 V for 30 min, followed by 170 V for 45 min. After Coomassie brilliant blue staining for 30 min, the samples were destained with 1% acetic acid until clear bands were visible. After imaging with a GenoSens 2000 gel imaging system, the grayscale value of each protein was analyzed using Image Lab software. The grayscale value of the protein at each 5 OD was measured. The whole-cell concentration was determined by controlling the protein ratio, and the amount of protein used was controlled by adjusting the cell concentration. The efficiency of spermidine synthesis in the surface display system was tested according to the conditions of Example 2.

[0089] Table 10. Whole-cell catalytic synthesis of spermidine at different protein expression levels Optimizing the proportions of whole-cell catalysts revealed that SCHSD and PcCASDH were used in greater quantities than CjCASDC1 in the spermidine biosynthesis pathway. This indicates that a sufficient substrate supply is a necessary prerequisite for constructing an effective whole-cell catalytic system. The efficiency of the whole-cell catalytic system reached a maximum of 18.84 g / L when the ratio of SCHSD1, PcCASDH1, and CjCASDC surface-displayed proteins was 3:2:1.

[0090] 2. Whole-cell catalytic fermentation temperature Cells A, B, and C were mixed in a protein ratio of 3:2:1 and the whole-cell catalysis was carried out under the whole-cell catalysis conditions described in Example 2, with the whole-cell transformation temperature controlled between 25°C and 40°C.

[0091] Table 11 Effect of different catalytic temperatures on whole-cell catalytic synthesis of spermidine As shown in Table 11, the constructed cells have wide adaptability at 25-40℃, which is beneficial for industrial application; at a temperature of 30℃, the yield of spermidine can reach up to 18.96 g / L.

[0092] Example 7 Comparison of intracellular enzyme expression and catalytic efficiency of whole-cell catalysts displayed on bacterial surface Using pET22b-schsd1-Ag43, pET22b-pccasdh1-Ag43, and pET22b-cjcasdc1-Ag43 as templates, the target fragments schsd1, pccasdh1, and cjcasdc1 were amplified using primers schsd1-F3 / R3, pccasdh1-F3 / R3, and cjcasdc1-F3 / R3. After purifying the fragments using a PCR product recovery kit, the target fragments were ligated into the linearized vector pRSFDuet from Example 5 according to the homologous recombination method in Example 1, and then transformed into the cloning host. E. coli DH5α competent cells were screened for correctly ligated recombinant plasmids pRSFDuet-schsd1, pRSFDuet-pccasdh1, and pRSFDuet-cjcasdc1 using the single colony screening and identification method described in Example 5, Section 1. Colony PCR primers were the universal pRSFDuet-1 primers T7 and T7t described in Example 5. Following the method described in Example 5, the successfully constructed recombinant plasmids were extracted and transformed into expression hosts. E. coli BL21 (DE3) competent cells, after colony PCR verification, yielded a host cell expressing intracellular proteins. E. coli / pRSFDuet-schsd1、 E. coli / pRSFDuet-pccasdh1 and E. coli / pRSFDuet-cjcasdc1.

[0093] Following the steps of induction expression in Example 1, whole cells expressing intracellular proteins were obtained, and then whole-cell catalysis was performed according to the protein usage ratio (3:2:1) and reaction temperature (30°C) optimized in Example 6.

[0094] Table 12 Sequence Information The results are as follows Figure 2 As shown, A represents the intracellular enzyme-expressed whole-cell catalyst, and B represents the bacterial surface-displayed whole-cell catalyst. When the whole-cell catalytic fermentation time was extended to 48 h, the whole-cell catalytic system composed of intracellular enzyme expression basically reached the highest efficiency of whole-cell catalysis, with a spermidine yield of 22 g / L; while the whole-cell catalyst based on bacterial surface display achieved a spermidine yield of 30 g / L after 48 h, and its yield continued to increase with time.

[0095] Example 8: Frequency of Reuse of Whole-Cell Catalyzed Spermine Batch-to-batch repeatability tests were conducted using the two whole-cell catalytic engineered bacteria and catalytic conditions described in Example 7. The whole-cell catalyst collected after the first whole-cell catalysis was centrifuged (5000 rpm, 5 min) and added to the second whole-cell catalysis system for a second batch repeat test. This process was repeated for the third and fourth batches.

[0096] Table 13 Catalytic results of reproducible experiments between different whole-cell batches The catalytic efficiency results are shown in Table 13. The whole-cell catalyst constructed based on bacterial surface display has a significantly higher batch-to-batch reusability efficiency than that of intracellular enzyme expression, which is more conducive to the recycling of engineered bacteria.

[0097] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. An enhanced surface-display engineered bacterium for whole-cell catalytic synthesis of spermidine, characterized in that, The enhanced surface-display engineered bacteria is *Escherichia coli*, which can display an enzyme protein that synthesizes spermidine on its surface. The enzyme protein is selected from at least one of homoserine dehydrogenase, carboxysemidine dehydrogenase, and carboxysemidine decarboxylase. The enzyme protein is expressed by fusion of a linker peptide with a carrier protein. Furthermore, the *E. coli* overexpresses an outer membrane protein. The amino acid sequence of the homoserine dehydrogenase is shown in SEQ ID NO:1 or 2; the amino acid sequence of the carboxysemine dehydrogenase is shown in SEQ ID NO:3 or 4; and the amino acid sequence of the carboxysemine decarboxylase is shown in SEQ ID NO:

5.

2. The enhanced surface-displaying engineered bacteria according to claim 1, characterized in that, The encoding nucleotide sequence of the homoserine dehydrogenase is shown in SEQ ID NO:11 or 12; the encoding nucleotide sequence of the carboxysemine dehydrogenase is shown in SEQ ID NO:13 or 14; and the encoding nucleotide sequence of the carboxysemine decarboxylase is shown in SEQ ID NO:

15.

3. The enhanced surface-displaying engineered bacteria according to claim 1, characterized in that, The amino acid sequence of the Linker peptide is shown in SEQ ID NO:

6.

4. The enhanced surface-displaying engineered bacteria according to claim 3, characterized in that, The nucleotide sequence of the Linker peptide is shown in SEQ ID NO:

16.

5. The enhanced surface-displaying engineered bacteria according to claim 1, characterized in that, The outer membrane protein is selected from a combination of BamA, BamB, BamC, BamD and BamE or Omp85; the carrier protein is Ag43 autotransporter.

6. The enhanced surface-displaying engineered bacteria according to claim 5, characterized in that, The outer membrane protein is Omp85; the amino acid sequence of Omp85 is shown in SEQ ID NO:7, 8 or 9; the amino acid sequence of the Ag43 autotransporter is shown in SEQ ID NO:

10.

7. The enhanced surface-displaying engineered bacteria according to claim 6, characterized in that, The nucleotide sequence encoding Omp85 is shown in SEQ ID NO:17, 18 or 19; the nucleotide sequence encoding Ag43 autotransporter is shown in SEQ ID NO:

20.

8. The enhanced surface-displaying engineered bacteria according to claim 1, characterized in that, An enzyme protein is a series of enzyme proteins linked together.

9. The enhanced surface-displaying engineered bacteria according to claim 8, characterized in that, Multiple enzyme proteins are the same enzyme protein.

10. The enhanced surface-displaying engineered bacteria according to claim 1, characterized in that, Multiple enzyme proteins are linked by a linker peptide; the amino acid sequence of the linker peptide is shown in SEQ ID NO:

6.

11. The enhanced surface-displaying engineered bacteria according to claim 1, characterized in that, Escherichia coli consists of Escherichia coli exhibiting homoserine dehydrogenase on its surface, Escherichia coli exhibiting carboxysemidine dehydrogenase on its surface, and Escherichia coli exhibiting carboxysemidine decarboxylase on its surface.

12. The enhanced surface-displaying engineered bacteria according to claim 11, characterized in that, The cell ratios of Escherichia coli exhibiting homoserine dehydrogenase, carboxysemine dehydrogenase, and carboxysemine decarboxylase were 1:1:1 to 3:3:2.

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